Bacterial vaginosis (BV) is the most common vaginal infection, affecting 29% of women of reproductive age, and is associated with major complications. Traditional tests for BV are inadequate. Newer commercial tests detect specific species or activities, but these are predicted to exclude many subgroups of BV patients. None of these tools adequately assess efficacy of treatment or even address recurrence. The proposed new diagnostic, Lactobacillus Relative Content (LbRC), has potential as a low cost, high throughput diagnostic and prognostic test for BV. The innovation of LbRC is that it uses a different strategy and tool to define the level of healthy of the vaginal microbiome, by measuring the relative amount of Lactobacillus to non- Lactobacillus with high sensitivity and providing an overview of what non-Lactobacillus groups are present. It is based on quantitative PCR (qPCR) using broad-spectrum bacterial 16S rRNA gene primers, and comparing two reactions, one with and one without inhibitors of Lactobacillus amplification (LB-blockers) (6). This reports both the titer of Lactobacillus relative to all non-Lactobacillus species and the overall composition of the various groups of non- Lactobacillus populations. We propose to validate LbRC in this project by showing that it correctly identifies known acute BV and healthy vaginal samples with high sensitivity and specificity. We further hypothesize that it can predict recurrence by its sensitive assessment of the initial post-treatment sample of BV patients, up to months before recurrence, and by identifying conversion (sweeping changes in vaginal bacteria) weeks before symptomatic BV recurs. Finally, we hypothesize that specific or groups of species identified in samples taken immediately before conversion may be responsible for conversion and ultimately BV. Our specific aims are: 1: Diagnosis. To determine its relative diagnostic potential, we will optimize and test LbRC to achieve >95% sensitivity and specificity of retrospectively and prospectively collected vaginal samples from women characterized as healthy or having acute BV, in side-by-side comparison to existing assays. 2: Prognosis. We will test whether LbRC, scored at either the conclusion of therapy or at our newly discovered event termed conversion, predicts recurrence of BV, and whether additional therapy at these time points delays or eliminates recurrence. 3. Causation. We will identify species prevalent in pre-conversion samples, using culture dependent and independent approaches, that may be responsible for conversion and ultimately for BV. Significance. Validation of LbRC in this project will pave the way for development of a superior and cost-effective clinical diagnostic/prognostic for BV for use in hospitals or clinical laboratories. The concept can then be developed into a POC or home-care device without using qPCR. LbRC will enable future testing and implementation of individualized treatment alternatives, to improve efficacy of therapies and prevent or delay recurrence. As a research tool, its identification of etiological agents of vaginal conversion may allow new approaches to therapy. By contributing to reduced incidence, duration, and recurrence of BV, LbRC will lower the large costs imposed by complications of BV.